1. Field of the Invention
The present invention generally relates to methods for calibrating clinical chemistry instruments.
2. Description of the Prior Art
Many clinical chemistry instruments measure optical changes caused by addition of a sample to test reagents. For example, some “dry chemistry” instruments measure reflectance factor changes after addition of a sample to a previously dry test slide. As a different example, some “wet chemistry” instruments measure transmittance changes after addition of a sample to a vessel, such as a cuvette, containing dissolved reagents. The sensitivity of either dry or wet chemistry instruments to measure optical changes in a sample-receiving reagent test slide or cuvette (or other sample container or liquid cell) may vary from instrument to instrument. Such instrument-to-instrument variations may result, among other things, from slight differences in the wavelength band shape or centroid of light emitted by a light source used in the optical system of the clinical instrument to illuminate the reagent test slide or cuvette.
Generally stated, to achieve consistency in optical measurements from instrument to instrument, calibration of each instrument is conventionally performed by running tests on the instrument with sample slides or cuvettes, comparing the measured test results with known reference or expected values, and adjusting the response of the instrument to compensate for the variability that may have arisen from, among other things, wavelength variabilities in the instruments' optical systems.
More specifically, control of the illumination band wavelength is important for accurate quantitation of dry chemistry test slides (or wet chemistry cuvettes). Wavelength control is substantially achieved by employing precision bandpass filters in the instruments' optical systems. However, even with precision control of the illumination or detection wavelength band, certain test assays, such as calcium, magnesium and albumin, may still require an offset to correct for differences in illumination wavelengths from instrument to instrument. For such chemistry slides or cuvettes, the reflection density or absorbance, respectively, bands are relatively narrow, where small deviations in the wavelength characteristics of the illuminating light could affect the sensitivity and accuracy of the instrument. Such assays (calcium, albumin and magnesium) are described in greater detail in the article, Dry Chemistry: Analysis with Carrier—Bound Reagents, authored by O. Sonntag, and published by Elsevier Science Publishers, Amsterdam, 1993, the disclosure of which is incorporated herein by reference. This problem of instrument-to-instrument variability in sensitivity is especially prevalent with test slides or cuvettes containing chromophores with a narrow reflection density or absorbance band or a steep baseline in the region of the band.
For example, in calcium dry reagent test slides, calcium ions complex with Arsenazo III, the reagent commonly used on such slides to quantify the calcium present in a blood, plasma, or serum sample under test, leading to a narrow absorbance band at approximately 680 nanometers. As can be seen from the graph shown in FIG. 4 depicting the reflectance spectrum of the Arsenazo III dye alone and after it is complexed with Ca2+ (calcium ion), the Arsenazo III reagent itself is strongly absorbing in most of the visible spectrum, with absorbance dropping off steeply from about 660 nanometers to about 740 nanometers. The Calcium-Arsenazo III complex's band is quite narrow, that is, about 40 nanometers full (band) width at half (band) maximum (FWHM), as can be seen from the difference of reflectance factor spectra, shown in FIG. 5. Maximum instrument sensitivity to calcium will be achieved when the complex's reflectance factor difference band centroid and shape are well matched to the illuminating light's band centroid and shape. In this example, it is assumed that the response of the detector of the instrument's optical system is flat over the wavelength range encompassing the narrow absorbance band of the calcium complex. However, the detector's response may also contribute to the overall sensitivity of the instrument if the detector's response is not substantially flat over the illumination wavelength range.
If the wavelength band of the slide illuminating light generated by the light source is not centered on the calcium complex's absorbance band, the sensitivity of the instrument to absorbance changes will be lower than it would be if the complex's absorbance and source's emission bands were better matched. If identical test samples were run on substantially identical calcium test slides, but on instruments with varying sensitivity to the complex, then otherwise uncompensated instruments with lower sensitivity would generally report lower calcium concentrations than those with higher sensitivity to the complex.
Likewise, and more commonly, if identical samples were run on substantially identical calcium test slides, but on instruments with varying and uncompensated baseline reflection densities or absorbances, the instruments with lower baselines would generally report lower calcium concentrations than those with higher baselines. Thus, a “calcium offset” is added to some clinical chemistry instruments' measurements to compensate for differences in the measured Arsenazo III baseline resulting from the variation in the illumination wavelength from instrument to instrument. This offset, as stated previously, is necessary to compensate for the very steep baseline in the region of the Calcium-Arsenazo III complex's absorbance peak.
The worse the instrument-to-instrument control of the illumination band wavelengths and band shape, the more instrument-to-instrument variability in uncompensated calcium concentration measurements (and to a lesser degree, other chemistries) is expected.
Some clinical chemistry instruments employ light emitting diodes (LEDs) as the illuminating light source. Most LEDs are substantially wavelength stable (minimal or negligible drift over time) and exhibit a relatively long life. While for some LEDs, the emission band shape and centroid (or peak) wavelength characteristics are well controlled, this is not the general case. Unfortunately, for currently available LEDs, the centroid wavelength (and band shape) of light frequency emission vary from LED to LED, and the centroid cannot be held to such close tolerances, for example, 680 nanometers plus or minus one Angstrom, to avoid this problem. Conventionally, bandpass filters are used, as mentioned previously, to control the illumination light's wavelength. Generally, bandpass filter specifications are more accurate and precise than LED band shape and peak wavelength specifications.
However, bandpass filters add complexity and cost to the clinical chemistry instrument's structure and operation. Optical filters can deteriorate over a relatively short period of time due to humidity and aging. (Most filters are guaranteed by the manufacturer for only one year, although advances in rugged bandpass filter manufacture typically extend their useful lifetime to several years.) Therefore, it is not uncommon that a clinical chemistry instrument must be periodically serviced to replace the filters, which sometimes cannot be done by the user. Such, of course, requires more downtime of the instrument and increased costs to the user or the manufacturer.
Furthermore, the bandpass filters must be precisely positioned with respect to the light source. Conventionally, such filters are positioned with respect to the light source such that the plane in which the filter resides is perpendicular to the optical axis of peak intensity. However, as disclosed in co-pending application Ser. No. 11/286,079 filed on Nov. 23, 2005, and entitled “Reflectometer and Associated Light Source For Use In a Chemical Analyzer”, the disclosure of which is incorporated herein by reference, it has been found that directing light from a light source, especially an LED, at an angle to the axis of peak intensity (that is, off-axis), to illuminate an object, such as a reagent test slide, creates a volume of homogeneous light which can solve problems associated with illumination homogeneity and Z-axis variability in the positioning of the object situated within that volume. But in this arrangement, the light from the LED sources is highly divergent and the illumination pathlengths are short. These angle-of-incidence and spatial constraints limit the effectiveness of using bandpass filters for the conditioning of the illumination band wavelengths.
Furthermore, calibrating each assembled clinical chemistry instrument by running performance tests using real reagent test slides is time consuming and presents difficulties. Instrument calibration using this conventional method may not be as precise as desired, since the offset adjustment of the instrument is at least partially based on performance tests using real test slides, making the results of this calibration also dependent on control sample preparation, stability, and handling, and also on the precision of the instrument measurements. Such variation may skew or increase the variability of the test results used in determining the offset to be applied to each instrument.